(1) To develop animal, especially nonhuman primate, models that mimic human disease: Over the past two years we have developed and characterized nonhuman primate disease models for two emerged respiratory viral pathogens; influenza A virus subtype H7N9 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Cynomolgus macaques were inoculated with A/Anhui/1/2013 (H7N9), a strain obtained from a fatal human case. Animals developed a transient, moderate pneumonia with virus replication occurring throughout the upper and lower respiratory tract. Analysis of gene expression profiles in lung lesions identified pathways involved in tissue damage and leads for therapeutics targeting host responses rather than virus replication. Disease models for MERS-CoV were developed and characterized in the rhesus macaque and common marmoset. The rhesus macaque mimics mild-moderate MERS in humans but not severe disease. This model has proven useful to assess vaccine efficacy (see below). Infection of the common marmoset led to the development of a progressive severe pneumonia with approximately 20% lethality. Extensive lesions and high viral loads were detected in the lungs and total RNAseq analysis demonstrated the induction of immune and inflammatory pathways. This model will in future be used for further drug efficacy testing. (2) To identify and characterize determinants of pathogenicity in animal models: Severe influenza virus infections are often associated with bacterial co-infections. This seems also be the case with the latest pandemic H1N1 virus. In order to study a potentiating effect of influenza and bacterial co-infection we performed a co-infection study in cynomolgus macaques using a moderately severe pandemic H1N1 strain (Ca04) and Methicillin-resistant Staphylococcus aureus (MRSA). Animals infected with MRSA only were largely asymptomatic, whereas animals infected with Ca04 only developed moderate pulmonary disease. Interestingly, animals initially infected with MRSA followed by Ca04 showed a dramatic reduction in clinical signs, whereas those initially infected with Ca04 followed by MRSA showed enhanced clinical disease (unpublished data). Similar studies were performed with a seasonal H3N2 virus and MRSA, in which we did not see disease reduction or enhancement. In response to the observation of a high prevalence of comorbidities in severe MERS-CoV cases, the effect of immunosuppression on outcome of MERS-CoV infection was tested in the Rhesus macaque model. Rhesus macaques were immunosuppressed through treatment with cyclophosphamide and dexamethasone before MERS-CoV inoculation. Immunosuppressed macaques did not develop more severe disease than immunocompetent animals, but they shed more virus, and viral loads in the lungs were significantly higher than in immunocompetent animals. Despite the increased virus replication, and in line with lack of increase in clinical disease, histological examination of the lungs showed a reduced inflammatory response in immunosuppressed macaques as compared to normal animals. These results suggested that the immune response to infection plays an important role in MERS-CoV pathogenesis (unpublished data). 3) To identify potential targets for intervention: For MERS, a first treatment study using a combination of ribavirin and interferon was performed in the rhesus macaque model showing efficacy as demonstrated by reduced clinical disease signs and viral load in lung tissue. This treatment scheme has been considered for human use in the Middle East endemic areas. In collaboration with the Molecular Targets Program at NCI, griffithsin, a novel viral entry inhibitor, was identified as having potent (EC50 5nM) activity against MERS-CoV. The post-exposure efficacy of nebulized griffithsin in the rhesus macaque model showed moderate reduction of viral load but did not significantly reduce disease signs (unpublished data). A pre-treatment study in rhesus macaques is scheduled. Additional antiviral drugs have also been tested in vitro with nitazoxanide showing a selectivity of approximately 10. An efficacy assessment in rhesus macaques is planned pending delivery of pharmacokinetic data in the rhesus macaque. We have also assessed other compounds in vitro including interferon-beta and Alferon (interferon alpha-n3) both of which have higher antiviral effect than interferon alpha2b. In addition, the cyclophilin inhibitors DEB025 and NIM811 were shown to have antiviral effect against MERS-CoV at clinically achievable concentrations. When DEB025 was combined with ribavirin the EC50 was reduced 4-fold suggesting that this combination may be suitable for use in humans. A highly potent near germ-line monoclonal antibody has also been assessed in the common marmoset model in a post-exposure setting. Treated animals showed little evidence of clinical signs compared to control animals; however, treated animals had surprisingly high viral loads at necropsy. To address this, a follow up pre-treatment study in marmosets is planned. (4) To develop vaccines and test their efficacy in the developed animal models: In order to develop a universal vaccine against influenza A viruses we are currently applying an approach based on two observations: i) highly conserved B cell epitopes are present within two separate helical regions within the hemagglutinin stalk region, and antibodies against them affords heterosubtypic binding and protection, and ii) removal of hemagglutinin globular region can increase antibody responses against other normally poorly antigenic epitopes. We used the Cytomegalovirus vector platform for these studies, which is able to induce long-lasting immune responses (both T cell and antibody) following a single vaccination. For MERS, we have tested a live-attenuated vaccine based on the vesicular stomatitis virus platform expressing the MERS-CoV spike protein. The vaccine reduced clinical disease and viral load and thus showed promising efficacy (unpublished data). We are currently testing a DNA vaccination platform expressing the MERS-CoV spike protein these studies are ongoing.